ApoE figures large in Alzheimer disease as the only known genetic risk factor for sporadic AD and an important regulator of amyloid-β (Aβ) metabolism and deposition (see ARF related news story). But for all the attention focused on this little protein, little is known about what normally controls the levels of ApoE in brain. In a paper published May 11 in the Journal of Biological Chemistry online, David Holtzman of Washington University in St. Louis and his colleagues show that the low-density lipoprotein receptor (LDLR) is the major receptor responsible for the for the uptake and degradation of ApoE-containing lipoproteins in murine brain. The researchers report that LDLR knockout mice had modestly increased extracellular ApoE in their brains and CSF, but the knockout did not affect amyloid plaque deposition when the mice were crossed with APP-transgenic mice. Using human ApoE knock-in mice, the researchers showed that LDLR also regulated CNS levels of the ApoE3 and 4 isoforms, but the possible impact of this increase on Aβ levels was not investigated.

To pinpoint which of several lipoprotein receptors are responsible for trafficking ApoE in the brain, first author John Fryer and his colleagues tested embryonic fibroblast cells from knockout mice for their ability to take up astrocyte-secreted ApoE-containing lipoprotein particles. Cells from LDLR knockout mice were unable to take up the lipoproteins, while fibroblasts from the low density lipoprotein receptor-associated receptor (LRP) knockout or wild-type mice took up the complexes avidly. CHO cells overexpressing LDLR, but none of four other candidate receptors, also took up and degraded astrocyte-derived ApoE, indicating that LDLR is the major, and perhaps only, receptor involved in clearing the types of ApoE-containing particles found in the brain.

Consistent with a lack of uptake of ApoE, the LDLR knockout mice had 50 percent more ApoE protein in the CNS and extracellular compartments in the brain. To see if this increase would be associated with higher Aβ, the researchers crossed the LDLR knockout mice with PDAPP mice. They saw no significant increases in either soluble or insoluble Aβ40 or Aβ42 in 3-month-old mice. In older animals (10 months), increases in plaque load and Aβ42 did not reach statistical significance. The mice had an increase in plasma cholesterol, but no changes in brain or CSF cholesterol, or APP levels or processing.

These results contrast with another knockout model where low expression of a different ApoE receptor, LRP, resulted in increased amyloid deposition in mice transgenic for human Aβ precursor protein (see ARF related news story). Fryer et al. speculate that perhaps the modest increase in ApoE seen in the LDLR knockouts is not sufficient to affect Aβ levels at the young ages or the early stages of deposition that were examined in this study. Or, LRP could be important for other, ApoE-dependent pathways that regulate Aβ metabolism or clearance.

The paper ends with a teaser. The authors present experiments that suggest the regulation of human ApoE protein levels differs significantly from that seen in mice. When they crossed mice carrying knocked-in human ApoE alleles with LDLR knockouts, offspring had two- to threefold higher levels of human ApoE3 and 4 in the CSF. It seems reasonable to ask whether this increase is of sufficient magnitude to enhance plaque deposition; the answer to that will require more experiments. However, to further muddy the waters, the authors report elsewhere (Fryer et al., 2005) that mating the human ApoE-expressing mice with APPsw mice yields animals with delayed Aβ deposition (see ARF related news story). Apparently, sorting out the role of the LDLR in regulating ApoE levels and in amyloid processing is going to take a few more crosses.—Pat McCaffrey

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  1. In a recent paper in JBC, Fryer and coworkers showed that the LDLR regulates ApoE levels in vitro and in vivo. The authors have previously shown that recombinant ApoE binds LRP but not LDLR. In a recent paper by Ruiz and coworkers (2005), the idea of receptor specificity was also raised, where LRP receptor, like LDLR, prefers lipid-bound forms of recombinant ApoE. However, here they show that LDLR, and not LRP or any other member of the LDL-receptor family, is responsible for astrocyte-secreted ApoE3 uptake and degradation. In addition, recombinant ApoE purified under reducing conditions was not a ligand for LRP, although it was a ligand for VLDLR.

    In LDLR-/- mice, murine ApoE levels are elevated both in CSF and in PBS extracted cortex. LDLR-/- X PDAPP(V717F) mice did not show any changes in either Aβ1-40 or 1-42 levels or deposition. However, CSF and cortical ApoE levels were not measured here. Previously, somewhat contrary reports of the role of E3 and E4 in Aβ clearance have been described. In a seminal study, Bales and coworkers demonstrated the ApoE-/- X PDAPP mice resulted in a significant decrease in amyloid burden (Bales et al., 1997). However, these mice crossed with GFAP-ApoE3 or E4 mice resulted initially in a further reduction of Aβ deposition (Holtzman et al., 1999), although animals aged beyond 12 months did exhibit Aβ deposition (ApoE4 > ApoE3) (Holtzman et al., 2000). A targeted replacement mouse expressing the different human ApoE isoforms (ApoE-TR), under the control of the endogenous mouse ApoE promoter, is perhaps the most relevant transgenic model available to study the effect of human ApoE isoform in the context of Aβ deposition. Here, ApoE-TR X LDLR-/- mice resulted in an increase in CSF ApoE3 and E4, almost to the level of ApoE2-TR mice.

    LDLR-/- mice are known to have significantly increased plasma cholesterol levels, but in the current paper, the authors reported no difference in hippocampal or CSF cholesterol. In the LDLR-/- X ApoE-TR mice, ApoE3 showed a trend toward reducing cholesterol levels in the cortex. Further study will be needed to determine if there is an ApoE isoform-specific “rescue” effect on CNS cholesterol levels in these mice.

    A more detailed study of the effect of LDLR on Aβ deposition and human ApoE isoform and cholesterol levels will be possible in an ApoE-TR X PDAPP/LDLR-/- mouse. It will also be necessary to further investigate the interaction between LDLR and LRP, as ApoE-mediated clearance of Aβ via LRP has previously been observed both in vitro and in vivo by the authors of the current paper.

    References:

    . Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition. Nat Genet. 1997 Nov;17(3):263-4. PubMed.

    . Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer's disease. J Clin Invest. 1999 Mar;103(6):R15-R21. PubMed.

    . Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2892-7. PubMed.

    . The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor. J Lipid Res. 2005 Aug;46(8):1721-31. PubMed.

  2. The study by Fryer et al. answers fundamental questions regarding the role of LDL receptor (LDL-r) family members in the regulation of apolipoprotein E (ApoE) in the brain [1]. Characterizing lipid physiology in the brain is critical for understanding Alzheimer disease (AD), given the role of ApoE4 as a risk factor for AD, the modulation of amyloid precursor protein (APP) metabolism by statin lipid-lowering drugs, and the involvement of ApoE and ApoE receptors in CNS injury and repair processes. Pitas first demonstrated that astrocytes take up ApoE incorporated in HDL-type particles via LDL-r [2], but the relative contributions of the other LDL-r family members LRP, VLDL-r, ApoER2, and gp330 have been less extensively studied. Using CHO cells transfected with the different ApoE receptors, Fryer demonstrated that only LDL-r significantly took up astrocyte-derived ApoE-containing lipoproteins. The physiologic relevance of LDL-r as the primary ApoE receptor in brain was supported by in vivo experiments. Mice lacking LDL-r accumulated higher levels of ApoE; when LDL-r-null mice were crossed with mice overexpressing different human ApoE isoforms, mice lacking LDL-r accumulated higher levels of ApoE3 and ApoE4, but not ApoE2, which binds poorly to the LDL-r.

    This paper complements a recent paper by Ruiz et al. in characterizing the binding specificity of ApoE to VLDL-r and LRP [3]. LDL-r requires lipid-bound ApoE and has a low affinity for ApoE2. Using in vitro binding assays, Ruiz demonstrated that LRP also preferred lipid-bound ApoE, but had affinity for all three ApoE isoforms. In addition to binding all isoforms of ApoE, VLDL-r also bound lipid-free ApoE. Together, these papers indicate that ApoE physiology is modulated by ApoE isoform, sialylation, disulfide dimerization, and lipid association that affects receptor interactions in the brain. The LDL-r receptor is the principal regulator of steady-state ApoE levels in the brain; since LRP and VLDL-r are upregulated in glia in CNS injury, it would be interesting to examine whether ApoE released in lesion models, for instance, is LRP or VLDL-r receptor-competent.

    References:

    . The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice. J Biol Chem. 2005 Jul 8;280(27):25754-9. PubMed.

    . Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta. 1987 Jan 13;917(1):148-61. PubMed.

    . The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor. J Lipid Res. 2005 Aug;46(8):1721-31. PubMed.

References

News Citations

  1. Lipoproteins and Amyloid-β—A Fat Connection
  2. It’s a RAP—Loss of LRP Increases Amyloid Deposition in Mice
  3. Sorrento: More Mice and Men—What Role ApoE in Vascular Dementia?

Paper Citations

  1. . Human apolipoprotein E4 alters the amyloid-beta 40:42 ratio and promotes the formation of cerebral amyloid angiopathy in an amyloid precursor protein transgenic model. J Neurosci. 2005 Mar 16;25(11):2803-10. PubMed.

Further Reading

No Available Further Reading

Primary Papers

  1. . The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice. J Biol Chem. 2005 Jul 8;280(27):25754-9. PubMed.